Crystalline dipeptides useful in the synthesis of elamipretide

ABSTRACT

Disclosed are crystalline forms of L-Lys(Boc)-Phe-NH2 and Boc-D-Arg-DMT.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Application No. 62/651,430, filed Apr. 2, 2018.

BACKGROUND OF THE INVENTION

Elamipretide (MTP-131) is a mitochondria-targeting peptide compound with therapeutic potential for treating diseases associated with mitochondrial dysfunction. Elamipretide contains four-amino acid residues and has been synthesized according to typical linear and convergent solution phase peptide synthesis methods. The synthetic routes to generate elamipretide that have been used to date require the preparation of various differentially protected peptides, such that certain protecting groups are selectively removed in order to subject the deprotected compound to peptide coupling, while other protecting groups remain to prevent unwanted side reactions. Even with protecting groups such coupling reactions and related steps generate impurities. Thus, there exists a need to develop new methods to purify elamipretide that allow the purification after coupling reactions. Crystallization of the desired reaction products are one method of achieving the necessary purification.

SUMMARY OF THE INVENTION

Disclosed are crystalline forms of L-Lys(Boc)-Phe-NH₂ and Boc-D-Arg-DMT, wherein DMT is an abbreviation for dimethyltyrosine, which are intermediates in the synthesis of elamipretide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the XRPD pattern of crystalline L-Lys(Boc)-Phe-NH₂.

FIG. 2 is the XRPD pattern crystalline Boc-D-Arg-DMT.

DETAILED DESCRIPTION OF THE INVENTION

Elamipretide has been shown to have various therapeutic effects in diseases related to mitochondrial dysfunction. Previous synthetic routes to elamipretide presented challenges with respect to scale-up due to reliance on chromatographic separations to enrich levels of desired intermediates. Herein are disclosed crystalline forms of L-Lys(Boc)-Phe-NH₂ and Boc-D-Arg-DMT, which can be used as purified intermediates in the synthesis of elamipretide.

One aspect of the present invention relates to crystalline forms of Compound (I):

which compound is also known as L-Lys(Boc)-Phe-NH₂.

A crystalline form of Compound (I) can be used in the synthesis of elamipretide.

In certain embodiments, a polymorph of the crystalline form is characterized by powder X-ray diffraction (XRD). θ represents the diffraction angle, measured in degrees. In certain embodiments, the diffractometer used in XRD measures the diffraction angle as two times the diffraction angle θ. Thus, in certain embodiments, the diffraction patterns described herein refer to X-ray intensity measured against angle 2θ.

In certain embodiments, a crystalline form of Compound (I) is not solvated (e.g., the crystal lattice does not comprise molecules of a solvent). In certain alternative embodiments, a crystalline form of Compound (I) is solvated. In some cases, the solvent is water.

In one aspect, the invention features a crystalline form of Compound (I) which has characteristic peaks in the powder X-ray diffraction (XRPD) pattern as shown in FIG. 1.

In another aspect, the invention features a crystalline form of Compound (I) which has characteristic peaks in the powder X-ray diffraction (XRPD) pattern at values of two theta (° 2θ) as shown in Table 1.

In another aspect, the invention features a crystalline form of Compound (I) which has characteristic peaks in the powder X-ray diffraction (XRPD) pattern at values of two theta (° 2θ) of: 4.7, 6.2, 12.4, 15.8, 16.5, 18.0, 18.2, 18.8, and 19.8.

In another aspect, the invention features a crystalline form of Compound (I) which has characteristic peaks in the powder X-ray diffraction (XRPD) pattern at values of two theta (° 2θ) of: 4.7, 6.2, 11.3, 12.4, 13.3, 15.0, 15.8, 16.5, 17.0, 17.7, 18.0, 18.2, 18.8, 19.8, 22.0, and 22.8.

The relative intensity, as well as the two theta value, of each peak in Table 1, as well as in FIG. 1, may change or shift under certain conditions, although the crystalline form is the same. One of ordinary skill in the art should be able to determine readily whether a given crystalline form is the same crystalline form as described in Table 1, as well as in FIG. 1, by comparing their XRPD data.

One aspect of the present invention relates to a crystalline form of Compound (II):

which compound is also known as Boc-D-Arg-DMT, and may also be drawn in the form of a zwitterion.

A crystalline form of Compound (II) can be used in the synthesis of elamipretide.

In certain embodiments, a polymorph of the crystalline form is characterized by powder X-ray diffraction (XRD). θ represents the diffraction angle, measured in degrees. In certain embodiments, the diffractometer used in XRD measures the diffraction angle as two times the diffraction angle θ. Thus, in certain embodiments, the diffraction patterns described herein refer to X-ray intensity measured against angle 2θ.

In certain embodiments, a crystalline form of Compound (II) is not solvated (e.g., the crystal lattice does not comprise molecules of a solvent). In certain alternative embodiments, a crystalline form of Compound (II) is solvated. In some cases, the solvent is water.

In one aspect, the invention features a crystalline form of Compound (II) which has characteristic peaks in the powder X-ray diffraction (XRPD) pattern as shown in FIG. 2.

In another aspect, the invention features a crystalline form of Compound (II) which has characteristic peaks in the powder X-ray diffraction (XRPD) pattern at values of two theta (° 2θ) as shown in Table 2.

In another aspect, the invention features a crystalline form of Compound (II) which has characteristic peaks in the powder X-ray diffraction (XRPD) pattern at values of two theta (° 2θ) of: 9.3, 12.1, 16.6, 17.6, 18.0, 18.8, and 19.4.

In another aspect, the invention features a crystalline form of Compound (II) which has characteristic peaks in the powder X-ray diffraction (XRPD) pattern at values of two theta (° 2θ) of: 9.3, 12.1, 13.7, 16.3, 16.6, 17.6, 18.0, 18.8, 19.4, 21.3, 23.0, 24.2, and 25.1.

The relative intensity, as well as the two theta value, of each peak in Table 2, as well as in FIG. 2, may change or shift under certain conditions, although the crystalline form is the same. One of ordinary skill in the art should be able to determine readily whether a given crystalline form is the same crystalline form as described in Table 1, as well as in FIG. 1, by comparing their XRPD data.

EXAMPLES

Materials and Methods

Name RM0858 General Method (2607) Parent 2Theta Sample Name S-18-0011882 SCC-169 File Name RM0858 General Method (2607) Scan Type Coupled TwoTheta/Theta Scan Mode Continuous PSD fast Start 2.000 End 40.016 Step Size 0.050 Time per Step 192.00 Anode Cu kα1 1.54 Generator kV 40.0 Generator mA 40.0 PSD Opening 2.940 Detector Slit Opening Primary Soller slit 2.500 Secondary Soller slit 2.500 Sample rotation speed 15.000 Divergence Slit 0.600 Antiscatter Slit 3.000 Slit Mode Fixed

Example 1. L-Lys(Boc)-Phe-NH₂ (Compound I)

Exemplary Synthetic Route

Synthesis of Compound 3

MW Molar Density BR Charge Name Amount (g) Volume (mL) (g/mol) Equiv Moles g/mL Amt (kg) kg/kg Phe-NH2 HCl 950.00 950.00 200.67 1.00 4.734 — 7.030 Cbz-Lys(Boc)-OH 1891.11 1891.11 380.44 1.05 4.971 — 13.990 1.99 HOBt-H₂O 797.48 797.48 153.14 1.10 5.208 — 5.900 0.84 Dimethylacetamide 3736.49 3987.71 87.12 — — 0.937 27.650 3.93 (DMAc) N-Methylmorpholine 957.72 1041.00 101.15 2.00 9.468 0.920 7.080 1.01 (NMM) EDCI 952.91 952.91 191.70 1.05 4.971 — 7.050 1.00 Anhydrous Ethyl 750.50 951.20 46.07 — — 0.789 5.550 0.79 Alcohol (EtOH) Acetonitrile (ACN) 11202.70 14252.80 84.93 — — 0.786 82.900 11.79 Acetonitrile (ACN) 11202.70 14252.80 85.93 — — 0.786 82.900 11.79

Operation Charge Units Inert the reactor with nitrogen. 1 Charge Compound 1 to reactor. 950.00 g 2 Charge Compound 2 to reactor. 1891.11 g 3 Charge HOBt-H2O to reactor. 797.48 g 4 Charge DMAc to reactor. 3736.49 g 5 Adjust solution to target 22° C. (19 to 25° C.) with agitation. 6 Agitate for 10-15 min at 22° C. (19 to 25° C.). 7 Slowly charge NMM to reactor with moderate 957.72 g agitation. 8 Adjust solution to target 7° C. (4 to 10° C.) with agitation. 9 Slowly charge EDCI to reactor with vigorous 952.91 g agitation. 10 Adjust solution to target 7° C. (4 to 10° C.) with vigorous agitation. 11 Charge EtOH to reactor with vigorous 750.50 g agitation. 12 Adjust solution to target 22° C. (19 to 25° C.) with vigorous agitation. 13 Agitate vigorously for ≥1 h at 22° C. (19 to 25° C.). IPC for Reaction Completion (≤1.0% Phe- 0.169% a/a NH2 remaining) CRYSTALLIZATION 15 Charge ACN to the reactor with vigorous 11202.70 g agitation. 16 Agitate vigorously for ≥5 h at 22° C. (19 to 25° C.). 17 Verify crystallization successful. 18 Filter the reaction to isolate the product (SCC-175). 19 Wash the product cake with ACN and combine 11202.70 g with mother liquor. 20 Dry the product with agitation and nitrogen bleed for at least 17 h.

Synthesis of Compound I

Volume MW Molar Density BR Name Amt (mL) (g/mol) Equiv g/mL Charge Amt units kg/kg Compound 3 2000.00 2000.00 526.63 1.00 15.700 kg 10% Pd/C 200.00 200.00 106.42 10% w/w — 3.140 kg 0.20 (50% w/w wet) Anhydrous Methyl 14191.08 17918.03 32.04 — 0.792 111.400 kg 7.10 Alcohol (MeOH) Anhydrous Methyl 7000.00 8838.38 32.04 — 0.792 54.950 kg 3.50 Alcohol (MeOH) Water 7000.00 7000.00 18.02 1.000 54.950 kg 3.50 Water 8000.00 8000.00 18.02 1.000 62.800 kg 4.00 MeOH-Water (1:9) 8000.00 7896.00 0.987 62.800 kg 4.00 Solution Water (1:9 MeOH- 9000.00 9000.00 18.02 1.000 NA NA water make-up) Anhydrous Methyl 792.00 1000.00 33.04 — 0.792 NA NA Alcohol (MeOH) (1:9 MeOH-water make-up)

Operation Charge Units REACTION (30 L Hydrogenator Main Reactor) Inert the hydrogenation reactor with nitrogen. 1 Charge Pd/C (10%, 50% w/w water, 20A597) to 200.00 g reactor. 2 Charge Compound 3 to reactor. 2000.00 g 3 Charge MeOH to the reactor. 14191.08 g 4 Adjust solution to target 22° C. (19 to 25° C.) with agitation. Inert the hydrogenation reactor with nitrogen. 5 Pressurize the reactor with hydrogen (20-25 psi). 6 Agitate for ≥6 h at 22° C. (19 to 25° C.) and 20-25 psi hydrogen. Note: Maintain the pressure at 20-25 psi hydrogen. 7 De-pressurize the reactor and inert with nitrogen at 22° C. (19 to 25° C.). IPC for Reaction Completion (≤0.5% SCC-175 0.09% a/a remaining) 9 Filter the reaction to remove catalyst. 10 Rinse the filter cake with MeOH and combine 7000.00 g with filtrate. DISTILLATION (30 L ChemGlass Jacketed Main Reactor) 11 Distill the reaction at ≤45° C. (100-200 Torr vacuum) to target. Note: Distillation target = 1.5-2.5 mL/g SCC- 5000.00 mL 175 charge. PRECIPITATION (30 L ChemGlass Jacketed Main Reactor) ISOLATION (12 L Allen Glass Filter w/30 micron ChemGlass teflon frit) 12 Charge water to reactor with moderate agitation 7000.00 g at 40° C. over 30-60 min. 13 Agitate the reaction at at 40° C. until crystallization observed. 14 Verify crystallization successful. 15 Charge water to reactor with moderate agitation 8000.00 g at 40° C. over 30-60 min. 16 Adjust the reaction to 22° C. (20 to 25° C.) over 30-60 min. 17 Filter the reaction to isolate the product (SCC- 169). 18 Wash the product cake with MeOH-water (1:9). 8000.00 g 19 Dry the product with agitation and nitrogen bleed for at least 17 h.

Preliminary Single-Solvent Solubility of Compound I:

Solvent Solubility (mg/mL)^(A) EtOH (SDAG-7) 154.8 THF 84.5 iPrOAc 5.1 MeOAc 18.2 Water 4.1 ^(A)Solubility was determined by HPLC (response curve).

Preliminary Precipitation Studies (MeOH/Water) of Compound I:

Precipitation Studies MeOH/ Addition Precip Concentration Solubility Experiment Water Mode (Y/N) (mL/g) (mg/mL) 1^(A) 1:3 Normal Y 10 7.0 2^(A) 1:5 Normal Y 10 5.0 3^(A) 1:9 Normal Y 10 3.6 4^(A)  1:19 Normal Y 10 2.7 5^(B) 1:3 Reverse N 80 — +2 parts 1:5 Normal Y 120 ND water ^(A)Experiments were conducted on 500 mg scale where the initial solution in MeOH (via 2447-41) was concentrated to a residue followed by charging MeOH/water at 25° C. ^(B)Experiment was conducted on 40 mg scale where the initial solution in MeOH (via 2447-41) was charged directly to water. An additional charge of water was required to enable precipitation.

In Process Precipitation Results (MeOH/Water) of Compound I:

Precipitation Results Concentration Solubility Experiment MeOH/Water (mL/g) (mg/mL) Total Losses 2447-41^(A) 1:9 13 2.5 5.7% 2447-47^(A) 1:5 10 4.2 6.2%

Example 2. Boc-D-ArmDMT-OH

Manufacturing Process to Produce Compound 8

Step Operation 1 Charge DMT-OBn HCl (1 eq) and Boc-D-Arg-OH HCl (1.05 eq) to the reactor. 2 Charge HOBt (0.1 eq) and DCM (8.5 L/kg of DMT-OBn HCl) to the reactor. 3 Adjust temperature to about 22 ± 3° C. and charge NMM (2.0 eq) to the reactor 4 Adjust temperature to about 15 ± 3° C. and charge EDCI (1.05 eq) to the reactor. 5 Adjust temperature to about 15° C. and agitate. 5 Charge EtOH (1.5 L/kg of DMT-OBn HCl) to the reactor 7 Agitate for a minimum of 5 hours at 15 ± 3° C. 8 Sample for in-process control reaction completion test, if reaction is not complete, charge additional EDCI, stir for a minimum of 1 hour andrepeat the in-process control test until criterion for completion is met. 9 Charge 15% EtOH in DCM. 10 Charge 1M HCl. 11 Adjust temperature to ambient and agitate. 12 Stop agitation and separate layers 13 Wash organic layer successively with brine, 1M HCl, brine and then brine again 14 Reduce volume via vacuum distillation 15 Adjust temperature to ambient 16 Charge EtOH and reduce volume via vacuum distillation 17 Sample for in-process control DCM content test, Repeat EtOH addition and volume reduction by vacuum distillation until the in-process control limit for DCM is met 18 Adjust temperature to ambient 19 Sample the product for in-process control test for SCC-192 purity and content

Manufacturing Process to Produce the HCl Salt of Compound II

Step Operation 1 Charge the ethanol solution of Boc-D-Arg-DMT-OBn HCl (SCC-192) 2 Charge 10% Pd/C, 50% wet (w/w) (20 wt. %) 3 Charge EtOH (7 L/kg of SCC-192) 4 Agitate and begin hydrogenation around ambient temperature 5 Step 2B In-Process Control 1: Test for reaction completion 5 Filter suspension through filter aid and wash filter cake three times with EtOH 7 Reduce volume via vacuum distillation 8 Adjust temperature to ambient 9 Charge THF and reduce volume via vacuum distillation 10 Adjust temperature to ambient 11 Charge THF and reduce volume via vacuum distillation 12 Adjust temperature to ambient 13 Charge THF and reduce volume via vacuum distillation 14 Step 2B In-Process Control 2: Test for EtOH content 15 Charge iPrOAc and agitate at ambient temperature 16 Filter and wash solids three times with iPrOAc 17 Dry the product under vacuum and nitrogen 18 Sample the product for in-process control test for purity 19 Step 2B In-Process Control 3: Test for Purity

Preparation of the Zwitterionic Form of Compound II

-   1. The crude HCl salt is suspended in CH₃OH/H₂O (1/1, v/v) -   2. The suspension is heated to 45˜50° C. -   3. After the clear solution is formed, an aqueous solution of Na₂CO₃     (1.2 eq.) is added. During the addition, the solid begins to     precipitate. -   4. The suspension is stirred for 1 h at 45° C., and then cooled to     15° C. and stirred for an additional hour. -   5. The solid is isolated by filtration and dried to provide Compound     II with high purity (99.4 area %) by HPLC. The calculated w/w assay     correcting for residual solvents, water content and residue on     ignition for this demonstration run was 98.7%.

Formation of zwitterionic compound led to high purity material that was stable, easily handled, and highly crystalline.

Example 3. Crystalline L-Lys(Boc)-Phe-NH₂—XRPD Peak List

Angle d Value Net Intensity Gross Intensity Rel. Intensity 4.704 18.77211 9559 11528 100.0% 5.230 16.88407 624 2301 6.5% 5.648 15.63394 862 2340 9.0% 6.200 14.24402 7663 8912 80.2% 6.650 13.28067 394 1456 4.1% 7.056 12.51772 508 1400 5.3% 7.709 11.45860 1161 1869 12.1% 8.553 10.33046 2135 2771 22.3% 9.402 9.39869 2473 3073 25.9% 9.922 8.90729 2627 3206 27.5% 10.303 8.57920 1435 1997 15.0% 11.292 7.82975 3301 3848 34.5% 11.755 7.52244 1985 2530 20.8% 12.265 7.24594 2827 3370 29.6% 12.452 7.10258 4581 5123 47.9% 12.655 6.98916 2764 3305 28.9% 13.270 6.66655 4117 4655 43.1% 13.778 6.42221 795 1330 8.3% 14.133 6.26164 333 866 3.5% 14.462 6.11961 882 1413 9.2% 14.957 5.91833 3726 4255 39.0% 15.756 5.61997 7404 7928 77.5% 16.263 5.44580 2360 2881 24.7% 16.504 5.36683 5366 5885 56.1% 16.961 5.22337 2930 3447 30.7% 17.107 5.17917 2585 3100 27.0% 17.658 5.01859 3807 4320 39.8% 17.956 4.93596 4360 4871 45.6% 18.210 4.86777 4451 4960 46.6% 18.832 4.70852 8963 9468 93.8% 19.561 4.53460 3331 3830 34.8% 19.802 4.47996 7877 8375 82.4% 20.265 4.37865 1989 2483 2.8% 20.715 4.28449 1746 2237 18.3% 21.514 4.12715 1756 2241 18.4% 21.958 4.04471 4087 4568 42.7% 22.807 3.89590 3856 4331 40.3% 23.398 3.79891 779 1248 8.1% 23.660 3.75740 835 1303 8.7% 24.306 3.65896 726 1188 7.6% 25.050 3.55198 2158 2614 22.6% 25.462 3.49548 764 1217 8.0% 25.709 3.46234 1227 1677 12.8% 26.016 3.42222 714 1161 7.5% 26.319 3.38357 521 966 5.5% 26.713 3.33445 662 1103 6.9% 27.411 3.25119 709 1143 7.4% 28.356 3.14493 831 1256 8.7% 28.808 3.09564 340 761 3.6% 30.662 2.91341 740 1142 7.7% 61.492 2.83854 649 1041 6.8% 35.876 2.50109 376 740 3.9% 36.369 2.46832 362 723 3.8%

Example 4. Crystalline Boc-D-Arg-DMT13 XRPD Peak List

Angle d Value Net Intensity Gross Intensity Rel. Intensity 8.253 10.70448 3779 4204 15.0% 9.353 9.44784 20863 21225 82.9% 12.104 7.30592 20016 20338 79.6% 12.809 6.90584 2797 3120 11.1% 13.755 6.43269 11432 11758 45.4% 15.709 5.53659 906 1235 3.6% 16.307 5.43127 7500 7829 29.8% 16.556 5.35006 12773 13103 50.8% 16.725 5.29645 2495 2825 9.9% 17.557 5.04740 22493 22824 89.4% 18.006 4.92236 25154 25485 100.0% 18.756 4.72740 18203 18534 72.4% 18.908 4.68953 4809 5141 19.1% 19.414 4.56848 12725 13056 50.6% 20.715 4.28417 899 1231 3.6% 21.259 4.17597 5674 5406 20.2% 22.357 3.97329 3830 4161 15.2% 23.009 3.86218 7511 7842 29.9% 23.153 3.83695 2203 2533 8.8% 23.511 3.78085 4659 4989 18.5% 24.208 3.57354 7257 7585 28.8% 24.809 3.58598 1940 2268 7.7% 25.060 3.55054 5627 5954 22.4% 25.767 3.45478 970 1297 3.9% 26.009 3.42308 1418 1744 5.6% 26.457 3.36613 255 580 1.0% 26.721 3.33347 161 485 0.6% 27.763 3.21973 1852 2185 7.4% 28.111 3.17181 1182 1503 4.7% 28.263 3.15505 366 687 1.5% 29.111 3.06507 2546 2864 10.1% 29.311 3.04456 1100 1418 4.4% 29.652 3.00931 616 933 2.4% 29.912 2.98475 1550 1865 6.2% 30.212 2.95583 1607 1922 6.4% 30.865 2.89475 1913 2226 7.6% 31.414 2.84540 1565 1876 6.2% 31.695 2.82084 468 778 1.9% 32.665 2.73926 657 963 2.6% 32.915 2.71901 296 602 1.2% 33.805 2.64939 326 632 1.3% 34.165 2.62230 670 976 2.7% 34.977 2.56330 485 795 1.9% 36.166 2.48167 798 1113 3.2% 36.514 2.45883 611 927 2.4% 36.813 2.43955 364 682 1.4% 37.466 2.39651 173 502 0.7% 37.966 2.36808 901 1257 3.6% 38.221 2.35285 394 764 1.6% 38.576 2.33202 878 1265 3.5% 39.114 2.30117 394 810 1.6% 39.516 2.27866 363 801 1.4%

INCORPORATION BY REFERENCE

All U.S. patents and U.S. and PCT published patent applications mentioned in the description above are incorporated by reference herein in their entirety.

EQUIVALENTS

Having now fully described the present invention in some detail by way of illustration and examples for the purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims. 

We claim:
 1. A crystalline form of Compound (I),

wherein said crystalline form has characteristic peaks in its XRPD pattern at values of two theta as described in Table
 1. 2. A crystalline form of Compound (I),

wherein said crystalline form has characteristic peaks in its XRPD pattern at values of two theta as described in FIG.
 1. 3. A crystalline form of Compound (I),

wherein said crystalline form has characteristic peaks in its XRPD pattern at values of two theta theta (° 2θ) of: 4.7, 6.2, 12.4, 15.8, 16.5, 18.0, 18.2, 18.8, and 19.8.
 4. The crystalline form of claim 3, wherein said crystalline form has characteristic peaks in its XRPD pattern at values of two theta (° 2θ) of: 4.7, 6.2, 11.3, 12.4, 13.3, 15.0, 15.8, 16.5, 17.0, 17.7, 18.0, 18.2, 18.8, 19.8, 22.0, and 22.8.
 5. A crystalline form of Compound (II),

wherein said crystalline form has characteristic peaks in its XRPD pattern at values of two theta as described in Table
 2. 6. A crystalline form of Compound (II),

wherein said crystalline form has characteristic peaks in its XRPD pattern at values of two theta as described in FIG.
 2. 7. A crystalline form of Compound (II),

wherein said crystalline form has characteristic peaks in its XRPD pattern at values of two theta theta (° 2θ) of: 9.3, 12.1, 16.6, 17.6, 18.0, 18.8, and 19.4.
 8. The crystalline form of claim 7, wherein said crystalline form has characteristic peaks in its XRPD pattern at values of two theta (° 2θ) of: 9.3, 12.1, 13.7, 16.3, 16.6, 17.6, 18.0, 18.8, 19.4, 21.3, 23.0, 24.2, and 25.1. 